Our research concerns the mechanism and consequences of Ty (Transposon yeast) element retrotransposition in the budding yeast Saccharomyces cerevisiae. Ty elements comprise five related families of long terminal repeat retrotransposons that transpose via an RNA intermediate. The Ty genome contains two genes, TYA and TYB, which correspond to the gag and pol genes of retroviruses, respectively. The retrotransposon is transcribed into a nearly genome-length RNA, which is the template for reverse transcription by the self-encoded reverse transcriptase protein and for translation. Ty protein maturation and reverse transcription take place within Ty virus-like particles (Ty-VLPs), which appear to be essential for the transposition process. Although Ty-VLPs accumulate in the cytoplasm, a Ty preintegration complex containing Ty cDNA, the element-encoded integrase and perhaps other proteins return to the nucleus, where integration takes place at different chromosome locations. We are particularly interested in the biology of Ty1 elements because these elements are the most abundant, competent for transposition, and their RNA transcripts accumulate to an exceptionally high level. Despite the abundance of Ty1 RNA, however, mature Ty1 proteins and VLPs are present at low levels, and Ty1 transposition events are also very rare. Although Ty1 elements preferentially integrate upstream of genes transcribed by RNA polymerase III, Ty1 insertions can mutate essentially any yeast gene, form large complex multimeric insertions of 100 kb or more, and can also initiate chromosomal deletions, inversions and translocations by homologous recombination with other Ty1 elements in the genome. Information gained from studying Ty elements has been successfully applied to several other areas of biomedical research. For example, understanding how Ty elements transpose in yeast has led to a greater understanding of how retroelements in other organisms including humans function, because many of these elements are related. Over 30% of the human genome is comprised of retroelement sequences, such as LINE and SINE, intracisternal A-type particle, and endogenous retroviral elements. Most importantly, genome rearrangements and insertional events involving these elements have been implicated in human disease and cancer. Completion of the human genome sequence coupled with further genomic analyses of cancerous cells will likely reveal new roles for retroelements that can be modeled in yeast using Ty elements or their mammalian counterparts. In addition, many aspects of the retrotransposon replication cycle are similar to those of retroviruses, including HIV. Therefore, steps in the process of retrotransposition can be compared and contrasted with similar processes in retroviruses to learn more about both classes of elements. Over the past year, we have made progress in the following areas. We, in collaboration with Dr. Robert Fisher (SAIC Frederick), have utilized a novel method for cleaving proteins with formic acid that is suitable for mass spectroscopy. Cleavage with formic acid is efficient and specific for aspartyl residues, and this specificity of cleavage lends itself easily to database searches. Parallel digests with trypsin suggest that formic acid cleavage generated comparable or better results than tryptic digestion for protein identification. We are currently using this technique to search for cofactors that associate with Ty-VLPs. We, in collaboration with an international consortium of yeast researchers headed by Dr. Mark Johnston (Washington University), have developed a near complete set (95% of all ORFs) of single gene deletions to systematically survey gene function. These mutations are currently being screened for their affects on Ty1 retrotransposition. In our continuing effort to identify cellular genes that modulate Ty1 retrotransposition, we have surveyed all members of the RAD2 family of nucleases for their affects on Ty1 retrotransposition. We have shown that only Rad27/Fen1, a highly conserved structure-specific nuclease important for DNA replication and genome stability, inhibits Ty1 mobility by affecting the fate of unincorporated cDNA.